Construction of the Recombinant Plasmid Expressing AID under the Control of Temperature-sensitive Promoter of Bacteriophage Lambda

Activation-induced cytidine deaminase (AID) is a B-cell specific enzyme responsible for somatic hypermutation (SHM) and class switch recombination (CSR) of antibody genes within the B-cell follicle of peripheral lymphoid organs. Ectopic overexpression of the enzyme leads to mutations in non-B cells and Escherichia coli (E.coli) genes. However, induction of mutations in E.coli by expression of AID requires highly regulated conditions. Therefore, the aim of this study was to construct a plasmid expressing AID under the control of tightly regulated temperature-sensitive promoter of bacteriophage lambda.. The AID gene was isolated from PGEMT recombinant plasmid, containing AID and cloned into PTG19-T vector. Then, AID was ligated to an expression plasmid under the control of left promoter of bacteriophage lambda and mutant thermolabile cI857 repressor. Finally, the resistance marker of the engineered plasmid was replaced by tetracycline resistance gene. The whole open reading frame of AID was ligated into a plasmid containing a cI857-sensitive receptor lambda bacteriophage. The sequence and reading frame accuracy of the cloning was confirmed by electrophoresis of PCR products on agarose gel, restriction enzyme digestion, and nucleotide sequencing methods. In current study, the AID was successfully cloned into an expression plasmid containing thermo-sensitive promoter of bacteriophage lambda. The recombinant plasmid could be used in different strains of E.coli to produce mutator strains.